England have released Dan Cole, Dan Robson, Joe Cokanasiga and Mike Brown to play for their clubs this weekend.
Cole is the only player of that quartet to have featured in both England’s first two Six Nations matches.
Centres Ben Te’o and Ollie Devoto, who have yet to play in this championship, are included in a 25-man squad for a three-day training camp this week.
England, who are top of the table after two wins, face Wales in Cardiff on 23 February.
Scrum-half Robson is set to play for Wasps at Bristol on Friday, while prop Cole is likely to be part of the Leicester team that play Saracens on Saturday.
Wing Cokanasiga should feature for Bath against Newcastle, while full-back Brown is clear to play when Harlequins host Worcester.
Forward Itoje has been ruled out of the Wales game after injuring a knee in the opening win over Ireland.
Co-captain Hartley has missed the first two matches with a knee injury.
On Tuesday, prop Mako Vunipola was ruled out of the rest of the Six Nations with an ankle problem.
Head coach Eddie Jones will name an updated squad on Sunday, before reducing it to 25 players on 19 February and finalising his starting XV and replacements on 21 February.
BBC Rugby correspondent Chris Jones
Leicester prop Dan Cole made his international return against France, but his exclusion from this training party suggests Harry Williams is in line to feature against Wales.
Joe Cokanasiga and Mike Brown have been unable to break into a matchday squad this Six Nations, with Elliot Daly, Jonny May, Chris Ashton and Jack Nowell currently the preferred options in the back three.
Dan Robson is being given game time with Wasps by the England management and is in line to be involved in Cardiff.
Ben Youngs, who has played all but 10 minutes of the Six Nations so far, is the only scrum-half in camp.
Ben Te’o missed England’s opener against Ireland through injury and was not selected for the meeting with France, but remains a key part of Jones’ plans.
England squad for training camp:
Luke Cowan-Dickie (Exeter Chiefs), Tom Curry (Sale Sharks), Ellis Genge (Leicester Tigers), Jamie George (Saracens), Nathan Hughes (Wasps), George Kruis (Saracens), Joe Launchbury (Wasps), Courtney Lawes (Northampton Saints), Brad Shields (Wasps), Kyle Sinckler (Harlequins), Billy Vunipola (Saracens), Ben Moon (Exeter Chiefs), Harry Williams (Exeter Chiefs), Mark Wilson (Newcastle Falcons).
Chris Ashton (Sale Sharks), Elliot Daly (Wasps), Ollie Devoto (Exeter Chiefs), Owen Farrell (Saracens), George Ford (Leicester Tigers), Jonny May (Leicester Tigers), Jack Nowell (Exeter Chiefs), Henry Slade (Exeter Chiefs), Ben Te’o (Worcester Warriors), Manu Tuilagi (Leicester Tigers), Ben Youngs (Leicester Tigers).